Project 2 Summary The immune system displays remarkable, layered complexity. Multiple cell types can adopt different effector programs?often based on the expression of one or more defining transcription factors?leading to diverse cell subtypes that have distinct functions. Emerging data reveals that after the polarized effector phase, heterogene- ity also develops among memory or memory-like populations. Though well known for T cells, this paradigm applies to B cells as well. Our group has defined several phenotypically and transcriptionally distinct MBC sub- sets with distinct functions in mice. During the first period of this project we have defined meaningful and repro- ducible cellular diversity within human MBC compartments. To do so we have used a combination of flow-based screening, RNA-seq, V-region high throughput sequencing (V-seq), and functional analysis to define the nature of MBCs across multiple tissues of the same individual and then over many different individuals. The functional significance of these cells and the heterogeneous expression of genes and surface markers on human MBC largely remain to be elucidated. Given our unique access via this PPG to both secondary lymphoid and mucosal tissues, a major focus of both this Project is resident MBCs in tissues (BRM). In recent work we have discovered surface markers and transcriptional profiles that define BRM. Using V region high-throughput sequencing (V- seq) we have further identified unexpectedly large clones of MBC and have tracked their migration patterns across tissues. These discoveries show the power of our approach and set the stage for deeper analysis to determine the functions of these newly identified cell types and clones, how they relate to each other, and what drives their expansion and differentiation. With this in mind, our Aims are: 1) To define novel subsets of BRM by single cell RNA-seq; deep RNA-seq and epigenetic analysis of purified populations; and by imaging. 2) To determine the functional responses of MBC subsets in tissues and SLT, testing the hypothesis that BRMs in mucosal sites include cells with innate-like qualities. 3) To define Ab networks and specificities of large B cell clones in different tissues. We will use deep V-seq to identify large clones among different MBC subsets and tissues to elucidate clonal flow and differentiation across MBC subsets and tissues. We will then express se- lected mAbs (derived from single cell HL sequences) from such large clones and screen them on self- and microbial- arrays to identify driving antigens. If we can find driving antigens for large clones, akin to how CMV and EBV expand CD8 cells, it would have major implications for understanding both microbiome-immune inter- actions and autoimmunity. Overall, these studies will provide important, useful and novel insights into how human B cell memory develops and how if functions at mucosal and lymphoid sites.